Quantification of PCR products by phosphate measurement.
Various techniques for quantification of PCR are available. Most frequently, the densitometric intensities of ethidium bromide-stained PCR products separated in gels are compared after normalizing to the levels of housekeeping gene products such as beta-actin. More precise, but extremely time consuming, is the technique of competitive PCR. Newer methods, such as tracking amplification in real-time, have high start-up and maintenance costs (e.g., TaqMan, Applied Biosystems; LightCycler, Roche; I-Cycler, Bio-Rad). Here, I describe an alternative, simple technique to quantify PCR products by determining the entire phosphate released during PCR. The method can be performed using common laboratory equipment, and the reagents needed are extremely cheap. The method is validated by measuring the induction of inducible nitric oxide synthase gene expression in cell culture and comparing the results with data obtained by LightCycler experiments and RNase protection assays.
Arneth
Borros
B
Institute for Clinical Chemistry and Laboratory Medicine, Johannes Gutenberg University Mainz, 55131 Mainz, Germany. arneth@zentrallabor.klinik.uni-mainz.de
D000818:Animals; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D004996:Ethidium / Q000737:chemistry; D051379:Mice; D010710:Phosphates / Q000032:analysis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D010710:Phosphates; D004996:Ethidium